Glut-1 translocation in FRTL-5 thyroid cells: role of phosphatidylinositol 3-kinase and N-glycosylation.

It was previously demonstrated that insulin or TSH treatment of FRTL-5 cells resulted in an elevation of glucose transport and in an increase of cell surface expression of the glucose transporter Glut-1. However, the signaling mechanisms leading to the insulin or TSH-induced increase in the cell surface expression of Glut-1 were not investigated. In the present study, we demonstrated that wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), interfere both in the signaling pathways of insulin and TSH leading to glucose consumption enhancement and Glut-1 translocation. Two hours after insulin treatment, TSH or cAMP analog (Bu)2cAMP stimulation, glucose transport was increased and most of the intracellular Glut-1 pool was translocated to plasma membranes. Wortmannin or LY294002 blocked the insulin, (Bu)2cAMP, and the TSH-induced translocation of Glut-1. Wortmannin or LY294002 alone did not alter the basal ratio between intracellular and cell surface Glut-1 molecules. These results suggest that in FRTL-5 cells wortmannin and LY294002 inhibited the insulin, (Bu)2cAMP and TSH events leading to Glut-1 translocation from an intracellular compartment to the plasma membrane. Likewise, (Bu)2cAMP effects on glucose transport and Glut-1 translocation to plasma membrane were repressed by PI3-kinase inhibitors but not by the protein kinase A (PKA) inhibitor H89. We suggest that (Bu)2cAMP stimulates Glut-1 translocation to plasma membrane through PI3-kinase-dependent and PKA-independent signaling pathways. To further elucidate mechanisms that regulate the translocation of Glut-1 to cell membrane, we extended this study to the role played by the N-glycosylation in the translocation and in the biological activity of Glut-1 in FRTL-5 cells. For this purpose we used tunicamycin, an inhibitor of the N-glycosylation. Our experiments with tunicamycin clearly showed that both the glycosylated and unglycosylated forms of the transporter reached the cell surface. Likewise, a decrease in glucose consumption (-50%) after treatment of cells with tunicamycin was accompanied by a decrease (-70% vs. control) in the membrane expression of a 50-kDa form of Glut-1 and an increase in its unglycosylated 41-kDa form. These results suggest that carbohydrate moiety is essential for the biological activity of glucose transport but is not required for the translocation of Glut-1 from the intracellular membrane pool to the plasma membrane.

Hexamethylene bisacetamide (HMBA) increases thyroglobulin levels in porcine thyroid cells without increasing cyclic-AMP.

The polar planar compound hexamethylene bisacetamide (HMBA) is an inducer of terminal differentiation which has been extensively studied in the murine erythroleukemia cells (MELC). We have tested this compound in normal porcine thyroid cells in primary culture where it either activates or inhibits the major tissue specific functions of these cells: it induces the reorganization of cells into follicles, prevents the loss of thyrotropin sensitivity in monolayer cells, activates cell growth but inhibits their iodide metabolism. In this paper, we demonstrate that HMBA acts on the total thyroglobulin levels measured in cell layers plus media. This specific marker of thyroid tissue is increased by HMBA both in kinetics and in concentration-response experiments. HMBA per se does not increase the total cyclic AMP measured either during the first hours after stimulation or in the following days when compared to controls. As expected, cyclic AMP in the same experiment increased rapidly within minutes after the cells were challenged by TSH (positive control). Altogether, the results show that the drug HMBA mimics thyrotropin effects on thyroglobulin levels measured in porcine thyroid cells in culture. This modulation cannot be explained by an increase in cyclic AMP, indicating that despite similarities between TSH and HMBA effects, the mechanism of the mode of action of these two molecules is very different.

Hexamethylenebisacetamide modulation of thyroglobulin and protein levels in thyroid cells is not mediated by phosphatidylinositol-3-kinase: a study with wortmannin.

Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action.

DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) increases iodide trapping, inhibits thyroperoxidase and antagonizes the TSH-induced apical iodide efflux in porcine thyroid cells.

4,4'-Di-isothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of several anionic channels and transporters including the band 3 protein of the red blood cell membrane was tested on iodide metabolism in cultured porcine thyroid cells. We used three experimental cell culture models: (i) forskolin-stimulated correctly inside-in polarized follicle-associated thyroid cells cultured onto plastic support (ii) suspensions of isolated cells derived from such cultures (iii) polarized monolayers in bicameral chambers. DIDS was observed to increase free-iodide trapping in all conditions. Organification of iodide by follicle-associated cell cultures incubated for 6 h decreased as a function of DIDS concentration with an IC50 of 5 x 10(-5) M. This block in organification is accounted for a block in thyroperoxidase activity as in vitro both purified lactoperoxidase and purified porcine thyroperoxidase were inhibited by DIDS with a similar dose-dependency the IC50 being also of 5 x 10(-5) M. Both control and DIDS-treated cells in suspension, actively trapped iodide and reached a steady concentration in about 50 min; however the plateau was 4.4-fold higher in (10(-3) M) DIDS-treated cells. Acute TSH-stimulation at this plateau of 125I-preloaded cells in suspension in the presence of 2 mM methimazole (MMI) induced a fast release of iodide from these cells as expected (first step of the TSH-biphasic effect). This TSH-induced iodide efflux was however completely inhibited by DIDS (10(-3) M). Furthermore, addition of DIDS to the apical compartment of TSH-prestimulated cell monolayers in bicameral chambers resulted in an increase in intracellular-iodide concentration and in an inhibition of iodide efflux into the apical medium. Taken together, the present results demonstrate that DIDS mainly interacts with two main components of the thyroid apical cell membrane: thyroperoxidase and a cAMP-sensitive iodide channel.

[Hexamethylene bisacetamide (HMBA) prevents the loss of porcine thyroid monolayer cells to thyrotropin sensitivity]. / L'hexaméthylène bisacétamide (HMBA) supprime la perte de sensibilité des cellules thyroïdiennes monocouches de porc à la thyrotropine.

The polar compound hexamethylenebisacetamide (HMBA) is a differentiating agent in the murine erythroleukemia cell system (MELC). It induces, like dimethylsulfoxide, the commitment to terminal differentiation leading to a recovery in the expression of several genes like the globin gene. This molecule which also induces differentiation in other cellular types is a growth agent for human, ovine and porcine thyroid cells. Forty-eight hours after the onset of culture, porcine thyroid monolayer cells do not respond to thyrotropin (TSH). We demonstrate that a pretreatment from the onset of culture with HMBA of porcine thyroid cells prevents the loss of TSH-sensitivity. The TSH-sensitivity is concentration-dependent in HMBA and leads to the reorganization of cells into follicles, even in the presence of HMBA. However, the withdrawal of HMBA when TSH is added is absolutely required to obtain a total recovery in iodide trapping and organification. If HMBA is present during TSH-stimulation, it inhibits iodide trapping partially but iodide organification completely Cells remain sensitive to TSH for at least 12 days if HMBA treated, and their sensitivity is totally restored after 3, 6 or 9 days of TSH-stimulation. HMBA, which is, like TSH, a growth agent for the thyroid cell and an agent that maintains some of the specialized functions, could be a putative candidate to obtain normal human thyroid cell lines.